Currently, there are four known hantaviruses associated with hemorrhagic fever with renal syndrome (HFRS): Hantaan virus (HTNV), Dobrava-Belgrade virus (DOBV), Puumala virus (PUUV), and Seoul virus (SEOV). Because distinct hantaviruses are usually carried by only one principal rodent host species, their distribution is generally limited to the range of that host (reviewed in Schmaljohn and Hjelle, 1997, Emerg. Infect. Dis. 3, 95-104). HTNV, carried by Apodemus agrarius, is found in Asia; DOBV, carried by Apodemus flavicollis, and PUUV, carried by Clethrionomys glareolus, are found in Europe. SEOV is more widely disseminated than any other recognized hantaviruses because its host, the common urban rat (Rattus norvegicus), is found throughout the world.
Puumala virus (PUUV), genus Hantavirus, family Bunyaviridae, is responsible for the vast majority of hemorrhagic fever with renal syndrome (HFRS) cases in Scandinavia, Europe, and Western Russia. In addition to the HFRS associated hantaviruses, there are also several hantaviruses in North, Central, and South America (e.g. Andes virus [ANDV]) that cause a vascular-leak disease known as hantavirus pulmonary syndrome (HPS) (Jonsson, C. B., Hooper J, Mertz G. Treatment of hantavirus pulmonary syndrome. Antiviral Research 2007 Nov. 21 Epub ahead of print). Hantaviruses are carried by persistently infected rodents and transmission to humans occurs when persons are exposed to rodent secreta or excreta. This usually occurs when persons inhale aerosolized rodent excreta. There is no Food and Drug Administration (FDA) approved vaccine or specific drug to prevent or treat HFRS or HPS.
Viruses in the Hantavirus genus (family Bunyaviridae) are enveloped and contain a genome comprised of three single-stranded RNA segments designated large (L), medium (M), and small (S) based on size (reviewed in Schmaljohn, 1996, In The Bunyaviridae Ed. R. M. Elliott. New York, Plenum Press p. 63-90). The hantavirus L segment encodes the RNA dependent RNA polymerase, M encodes two envelope glycoproteins (G1 and G2, also known as Gn and Gc), and S encodes the nucleocapsid protein (N).
A number of inactivated HFRS vaccines derived from cell culture or rodent brain were developed and tested in Asia (Lee et al., 1990, Arch. Virol., Suppl. 1, 35-47; Song et al., 1992, Vaccine 10, 214-216; Lu et al., 1996, J. Med. Virol. 49, 333-335). Drawbacks of these traditional killed-virus vaccines include a requirement for appropriate containment for the growth and manipulation of virus, and the necessity to ensure complete inactivation of infectivity without destroying epitopes on the virion important for protective immunity. In order to overcome these drawbacks, vaccine approaches involving recombinant DNA technology were developed including: vaccinia-vectored vaccines (Schmaljohn et al. 1990, J. Virol. 64, 3162-3170; Schmaljohn et al. 1992, Vaccine 10, 10-13; Xu et al. 1992, Am. J. Trop. Med. Hyg. 47, 397-404), protein subunit vaccines expressed in bacteria or insect cells (Schmaljohn et al. 1990, supra; Yoshimatsu et al., 1993, Arch. Virol. 130, 365-376; Lundkvist et al., 1996, Virology 216, 397-406), and a hepatitis core antigen-based recombinant vaccine (Ulrich et al., 1998, Vaccine 16, 272-280). For a review of hantavirus vaccine efforts see the review by Hooper and Li (Hooper and Li, 2001).
Vaccination with vaccinia recombinants expressing the M segment of either HTNV or SEOV elicited neutralizing antibodies and protected rodents against infection with both HTNV and SEOV, suggesting that an immune response to G1-G2 alone can confer protection (Schmaljohn et al. 1990, supra; Xu et al. 1992, supra; Chu et al. 1995, J. Virol. 69, 6417-6423). Similarly, vaccination with G1-G2 protein expressed in insect cells (baculovirus recombinant virus system) elicited neutralizing antibodies and protected hamsters from infection with HTNV (Schmaljohn et al. 1990, supra). In both the vaccinia and baculovirus systems, vaccination with G1-G2 provided more complete protection than G1 or G2 alone (Schmaljohn et al. 1990, supra). There are reports that candidate DNA vaccines comprised of around 500 nucleotide stretches of the Sin Nombre virus (SNV) M gene, or the full-length S gene, are immunogenic in mice (Bharadwaj, et al., 1999, Vaccine 17, 2836,43) and conferred some protection against infection with SNV in a deer mouse infection model (Bharadwaj, et al., 2002, J. Gen. Virol. 83, 1745-1751). The protection was surmised to be cell-mediated because there was no convincing evidence that these constructs elicited a neutralizing, or otherwise protective, antibody response. There have been several publications reporting the successful use of plasmid DNA vaccines containing the full-length M gene of SEOV, HTNV, ANDV, including the following reports:    1. Hooper, J. W., K. I. Kamrud, F. Elgh, D. Custer, and C. S. Schmaljohn (1999). DNA vaccination with hantavirus M segment elicits neutralizing antibodies and protects against Seoul virus infection. Virology, 255:269-278.    2. Hooper, J. W., D. Custer, E. Thompson, and C. S. Schmaljohn (2001). DNA Vaccination with the Hantaan virus M gene protects hamsters against three of four HFRS hantaviruses and elicits a high-titer neutralizing antibody response in rhesus monkeys. Journal of Virology 75:8469-8477.    3. Custer, D. M., E. Thompson, C. S. Schmaljohn, T. G. Ksiazek, and J. W. Hooper (2003). Active and passive vaccination against hantavirus pulmonary syndrome using Andes virus M genome segment-based DNA vaccine. Journal of Virology 79:9894:9905.    4. Hooper, J. W., D. M. Custer, J. Smith, and Victoria Wahl-Jensen. Hantaan/Andes virus DNA vaccine elicits a broadly cross-reactive neutralizing antibody response in nonhuman primates (2006). Virology 347:208-216.
In all cases high titer neutralizing antibodies were detected in animals (including nonhuman primates) vaccinated with the full-length M gene DNA vaccines, and protection from infection was achieved in rodent models. Neutralizing antibody responses to G1-G2 in the aforementioned vaccine studies correlated with protection, suggesting that neutralizing antibodies not only play an important role in preventing hantavirus infection, but also might be sufficient to confer protection. Passive transfer of neutralizing monoclonal antibodies (MAbs) specific to either G1 or G2 protected hamsters against HTNV infection (Schmaljohn et al., 1990, supra; Arikawa et al., 1992, J. Gen. Virol. 70, 615-624), supporting the idea that neutralizing antibodies alone can confer protection. This is further supported by the finding that serum from nonhuman primates vaccinated using a gene gun with DNA vaccines containing the HTNV or ANDY full-length M genes protected hamsters from infection with HTNV or lethal disease caused by ANDV Custer, D. M., E. Thompson, C. S. Schmaljohn, T. G. Ksiazek, and J. W. Hooper (2003). Active and passive vaccination against hantavirus pulmonary syndrome using Andes virus M genome segment-based DNA vaccine. Journal of Virology 79:9894:9905). Similarly, sera from rabbits vaccinated with the ANDV M gene-based DNA vaccine using electroporation protected hamsters from a lethal challenge with ANDV (Hooper J. W., A. M. Ferro, and V. Wahl-Jensen. Immune Serum Produced by DNA Vaccination Protects Hamsters Against Lethal Respiratory Challenge with Andes Virus (2008). Journal of Virology 82:1332-1338.)
Like all members of the Bunyaviridae, PUUV are enveloped viruses with tri-segmented (S, M, and L), negative sense, RNA genomes. The M genome segment encodes the Gn and Gc surface glycoproteins. These proteins are the targets of neutralizing antibodies found in the convalescent serum of patients and infected animals. Hitherto, attempts to produce vaccines that produce neutralizing antibodies against PUUV have been unsuccessful.
Thus, there is currently no vaccine against hemorrhagic fever with renal syndrome (HFRS) caused by Puumala virus. Killed PUUV vaccine approaches have not resulted in a useful, licensed product anywhere in the world. Molecular vaccines using the PUUV N protein have been tested in animal infection models and there have been reports of protection in the absence of neutralizing antibodies. (See Hooper and Li, 2001) Whether an N protein-based PUUV will be effective in species other than mice remain unknown. Efforts to develop molecular vaccines that elicit neutralizing antibodies against PUUV have been ongoing for several years without success. Attempts to use HTNV, SEOV, and/or ANDV M gene-based DNA vaccines to produce antibodies to cross-neutralize PUUV have resulted in only very low levels of cross-neutralization, and no protection (see Hooper 2001 and Hooper 2006 referenced above). It is extremely difficult to construct a plasmid that is actually capable of eliciting good neutralizing antibody responses against PUUV, probably due at least in part to the instability of the full-length M gene in E. coli plasmid systems. This barrier has been overcome by the invention described herein.
In previous attempts, the inventor produced a version of a Puumala virus M gene-based DNA vaccine (designated pWRG/PUU-M-(x22), described below) that was capable of eliciting antibodies in monkeys after several vaccinations, but acceptable levels of neutralizing antibodies were not produced. Moreover, pWRG/PUU-M-(x22) was not immunogenic in hamsters. And finally, pWRG/PUU-M-(x22) was unstable during plasmid amplification in E. coli due to undefined properties of the M gene sequence.
The inventor is named as an inventor on other U.S. patents related to vaccines for hantaviruses and poxviruses, namely U.S. Pat. No. 6,451,309; U.S. Pat. No. 6,620,412; U.S. Pat. No. 6,562,376 and U.S. Pat. No. 7,217,812. The entire contents of these patents are incorporated herein by reference.